CN115536747B

CN115536747B - Antibody combined with TROP2, bispecific antibody targeting TROP2 and CD3, and preparation method and application thereof

Info

Publication number: CN115536747B
Application number: CN202110736045.5A
Authority: CN
Inventors: 袁清安; 白丽莉; 孟庆武; 李艺佳; 赵立坤; 李延虎
Original assignee: Excyte Beijing Pharmaceutical Technology Development Co ltd
Current assignee: Excyte Beijing Pharmaceutical Technology Development Co ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2024-08-27
Anticipated expiration: 2041-06-30
Also published as: CN115536747A; WO2023273595A1

Abstract

The invention relates to the technical field of genetic engineering antibodies, in particular to an antibody combined with TROP2, a bispecific antibody targeting TROP2 and CD3, a preparation method and application thereof. The TROP2 antibody provided by the invention has good capability of combining TROP2 and high affinity; the bispecific antibody combined with TROP2 and CD3 has excellent biological functions of anti-TROP 2 and anti-CD 3 antibodies, can build a bridge between tumor cells and immune effector cells, effectively activate the immune effector cells and guide immune reaction, remarkably enhance the effect of killing the tumor cells by the immune cells, simultaneously furthest reduce ADCC effect, has higher safety and has better application prospect in the immunotherapy of tumors.

Description

Antibody combined with TROP2, bispecific antibody targeting TROP2 and CD3, and preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering antibodies, in particular to an antibody combined with TROP2, a bispecific antibody targeting TROP2 and CD3, a preparation method and application thereof.

Background

The antibody medicine is a biological macromolecular medicine prepared by antibody engineering technology taking cell engineering technology and genetic engineering technology as main bodies, and has the advantages of high specificity, uniform property, directional preparation aiming at specific targets and the like. Monoclonal antibodies are used clinically mainly in three ways: tumor treatment, immune disease treatment, and anti-infective treatment. Among them, tumor treatment is the most widely used field of monoclonal antibodies at present, and the number of products for tumor treatment is about 50% among monoclonal antibody products which have been put into clinical trials and marketed at present. Monoclonal antibodies are an immunotherapy for stimulating the immune system to kill target cells aiming at specific targets of lesion cells, and various methods are tried to modify antibody molecules in order to enhance the effector functions of the antibodies, in particular to improve the effect of killing tumor cells.

There are various methods for obtaining specific antibodies. The traditional hybridoma technology is to immunize a mouse or a rat, fuse spleen cells with myeloma cells, then dilute (to one cell per hole) and culture, detect the combination of hybridoma cell supernatant and immune antigen by an enzyme-linked immunosorbent assay (ELISA), and further screen monoclonal cell strains capable of secreting antibodies specifically combined with the antigen. If the murine antibody is applied to clinical drug development, the deimmunization modification is required by genetic engineering means such as hybrid antibody technology (chimerization) and humanized technology (humanization). In order to eliminate the immunogenicity problem that must be solved in the future clinically from the initial stage, the transgenic murine technology (TRANSGENIC MOUSE) can obtain fully human monoclonal antibodies by replacing the murine variable region with a human variable region so that the variable region sequence of the obtained monoclonal antibody is mainly human, and then further replacing the constant region. Another technique is phage surface display (PHAGE DISPLAY) technique. Phage surface display technology is the construction of engineered antibodies, typically in single chain or Fab format, in test tubes. However, the gene fragments constituting the engineered antibodies are derived from the gene bank of human immune cells, so that the obtained antibodies are fully human. Antibodies obtained by a number of phage technology have entered clinical studies or been approved for disease treatment in various ways.

On the basis of monoclonal antibodies, in order to further improve the drug effect and reduce the toxic and side effects, novel antibody drugs based on monoclonal antibodies, including drug-coupled antibodies (Antibody drug conjugate, ADC), bispecific antibodies (bispecific antibody, bsAb), CAR-T (CHIMERIC ANTIGEN receptor T cells) and other novel proteins and cell drugs, are rapidly developed. The cell-bridging double antibody based on the double specificity construction has the capability of connecting immune T cells and cancer target cells, so that the killing activity and specificity are obviously improved, and the double antibody has breakthrough clinical value and receives more attention.

Bispecific antibodies can be obtained in a variety of ways, and are prepared by a process that involves: chemical coupling methods, hybrid-hybridoma methods, and genetically engineered antibody preparation methods. The chemical coupling method is to link 2 different monoclonal antibodies together by chemical coupling, and the bispecific monoclonal antibody is prepared, which is the earliest bispecific monoclonal antibody. The hybrid-hybridoma method generates bispecific monoclonal antibodies by means of cell hybridization or ternary hybridomas obtained by established hybridoma fusion or established hybridoma fusion with lymphocytes from mice, and is therefore limited in its use to the production of bispecific antibodies of murine origin. With the rapid development of molecular biology technology, various construction modes of genetically engineered humanized or fully human bispecific antibodies appear, mainly comprising four classes of bispecific minibodies, diabodies, single chain diabodies and multivalent bispecific antibodies. At present, a plurality of genetically engineered bispecific antibody medicaments enter a clinical test stage and have good application prospect.

Trophoblast cell surface antigen 2 (TROP 2) is a cell surface glycoprotein expressed by the TACSTD gene encoding, also known as tumor-associated calcium signaling transducer 2 (TACSTD), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA 733-1), surface marker 1 (M1S 1), cell surface glycoprotein identified earlier than human placental trophoblasts, was later found to be highly expressed in most human solid tumor cancer cells and limited or restricted to normal human tissue. TROP2 belongs to the GA733 protein family, and has higher structural sequence similarity with epithelial cell adhesion molecules (EpCAM, also called TROP1 and TACSTD 1) and homology of 49 percent. TROP2 is composed of 323 amino acids, wherein the signal peptide contains 26 amino acids, 248 amino acids in the extracellular region, 23 amino acids in the transmembrane region and 26 amino acids in the cytoplasmic region, and the structure diagram is shown as A in FIG. 1. TROP2 promotes tumor cell growth, proliferation and metastasis mainly by modulating calcium ion signaling pathways, cyclin expression, and reducing fibronectin adhesion. TROP2 can also interact with β -catenin in the Wnt signaling cascade and thus act on transcription of nuclear oncogenes and proliferation of cells. Numerous clinical studies and literature reports indicate that TROP2 is overexpressed in epithelial cancers such as breast, pancreatic, gall bladder, colon, gastric, non-small cell lung, prostate, uterine and oral squamous carcinoma, while being rarely or not expressed in normal tissues of adults; overexpression of TROP2 in tumor tissue is closely related to poor prognosis and metastasis of cancer cells in patients, while affecting overall survival in patients. Thus, TROP2 has become an attractive target in tumor molecular targeted therapies.

TROP2 is a transmembrane protein, whose extracellular domain is widely distributed over a variety of tumor cells, and thus becomes a natural candidate for targeted therapy. Tissue expression limitations of TROP2 lead to reduced toxicity of the treatment, which is also an advantage of targeted TROP2 treatment. Antibodies, antibody conjugates, combinations, and other forms of drug targeting TROP2 are under development. The utility of anti-TROP 2 antibodies coupled to other chemotherapeutic agents has been demonstrated in various preclinical studies. Antibody-conjugated drug (ADC) IMMU-132 for the treatment of TROP2 overexpressed epithelial malignancy has been approved by the FDA for marketing (month 4 of 2020). The novel antibody coupling drug Sacituzumab govitecan (IMMU-132) is formed by coupling a humanized antibody hRS7 serving as a targeting vector with an irinotecan Kang Huoxing metabolite SN38 by taking TROP2 as a target point, and can be used for treating various epithelial malignant tumors such as breast cancer (triple negative breast cancer), ovarian cancer, small cell lung cancer and the like. In addition, other humanized anti-TROP 2 IgG-SN-38 conjugates, such as anti-TROP 2 hRS7-CL2A-SN-38 antibody conjugated drugs, have been demonstrated to have significant specific anti-cancer effects in xenograft models of various tumor cell lines (Calu-3, capan-1, bxPC-3, and COLO-205). In view of the above, it is important to develop a specific antibody against TROP2, in particular a fully humanized anti-TROP 2 antibody with good antigen binding properties to TROP 2.

The CD3 molecule on the surface of T cells consists of 4 subunits δ, ε, γ, ζ, whose molecular masses are 18.9k Da,23.1kDa,20.5kDa and 18.7kDa, respectively, whose lengths are 171, 207, 182, 164 amino acid residues, which together form 6 peptide chains, which are often tightly bound to a T Cell Receptor (TCR) to form a TCR-CD3 complex containing 8 peptide chains, the schematic structure of which is shown in fig. 1B. The complex has the functions of T cell activation, signal transduction and stabilization of TCR structure. The cytoplasmic segment of CD3 contains the immunoreceptor tyrosine activation motif (immunoreceptor tyrosine-based activation motif, ITAM), and TCR recognizes and binds to an antigenic peptide presented by MHC (major histo-compatibility complex) molecules, resulting in the phosphorylation of tyrosine residues in the conserved sequence of the ITAM of CD3 by the T-cell tyrosine protein kinase p56lck, which then recruits other SH2 (Scr homolog 2) domain-containing tyrosine protein kinases (e.g., ZAP-70). Phosphorylation of ITAM and binding to ZAP-70 are one of the important biochemical reactions in the early stages of the T cell activation signaling process. Thus, the function of the CD3 molecule is to transduce an activation signal generated by the TCR recognition antigen. Based on the function of CD3, it is important to develop a bispecific antibody that can be used for immunotherapy, binding both TROP2 and CD 3.

Disclosure of Invention

The first object of the present invention is to provide an antibody that binds to TROP2 and an active fragment thereof.

A second object of the present invention is to provide bispecific antibodies and active fragments thereof that bind TROP2 and CD 3.

It is a third object of the present invention to provide nucleic acids encoding TROP2 antibodies, bispecific antibodies that bind TROP2 and CD 3.

A fourth object of the present invention is to provide the use of a TROP2 antibody as described above, a bispecific antibody binding TROP2 and CD3 or an active fragment thereof.

Specifically, the invention provides the following technical scheme:

First, the present invention provides a TROP2 antibody comprising a heavy chain variable region and a light chain variable region, wherein CDR1, CDR2, CDR3 of the heavy chain variable region have the amino acid sequences shown in SEQ ID nos. 1 to 3, respectively, or have the amino acid sequences comprising a combination of one or more of the following mutations with the amino acid sequences shown in SEQ ID nos. 1 to 3 as reference sequences, respectively:

(1) S at position 1 of the amino acid sequence shown in SEQ ID NO.1 is mutated into N;

(2) The 4 th P mutation of the amino acid sequence shown in SEQ ID NO.2 is R, K or G;

(3) The P mutation at position 1 of the amino acid sequence shown in SEQ ID NO.3 is H, A;

(4) The N at the 2 nd position of the amino acid sequence shown in SEQ ID NO.3 is mutated into E;

The CDR1, CDR2 and CDR3 of the light chain variable region respectively have the amino acid sequences shown in SEQ ID NO.4-6 or respectively have the amino acid sequences which take the amino acid sequences shown in SEQ ID NO.4-6 as reference sequences and contain one or a combination of the following mutations:

(1) The 5 th G of the amino acid sequence shown in SEQ ID NO.4 is mutated into N or E;

(2) S mutation at position 7 of the amino acid sequence shown in SEQ ID NO.4 to R or K

(3) The 1 st A of the amino acid sequence shown in SEQ ID NO.5 is mutated into R;

(4) The S mutation at the 3 rd position of the amino acid sequence shown in SEQ ID NO.5 is G, H or R;

(5) The S at position 4 of the amino acid sequence shown in SEQ ID NO.5 is mutated to K.

The invention screens out the gene engineering single-chain antibody of anti-TROP 2 from the total synthetic single-chain human antibody library, obtains the variable region sequence of the antibody, constructs a mutation library through a point mutation kit, obtains clones with high affinity, combines the cloned DNA, assembles the single-chain antibody combined library in a recombination mode, and obtains the TROP2 antibody with high affinity combined with human TROP2 after screening.

The amino acid sequence mode of the antibody variable region provided by the application is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In the present application, the region division of FR and CDR is based on Kabat naming system. Here, FR 1-4 represent 4 framework regions and CDR 1-3 represent 3 hypervariable regions. FR 1-4 may be isolated from constant region sequences (such as the most common amino acids of the human immunoglobulin light and heavy chain class, subclass, or subfamily), isolated from human antibody frameworks alone or combined from different framework region genes.

Further, the present invention provides a TROP2 antibody comprising a heavy chain variable region and a light chain variable region, wherein CDR1 of the heavy chain variable region has the amino acid sequence shown in any one of SEQ ID nos. 1, 7, CDR2 has the amino acid sequence shown in any one of SEQ ID nos. 2, 8, 9, and CDR3 has the amino acid sequence shown in any one of SEQ ID nos. 3, 10;

CDR1 of the light chain variable region has any one of the amino acid sequences shown in SEQ ID No.4, 11 and 12, CDR2 has any one of the amino acid sequences shown in SEQ ID No.5, 13 and 14, and CDR3 has any one of the amino acid sequences shown in SEQ ID No.6 and 15.

Further screening, the invention provides a TROP2 antibody having a higher binding affinity for TROP2, wherein the CDRs of the heavy chain variable region are any one of:

(1) CDR1 has the amino acid sequence shown as SEQ ID NO.1, CDR2 has the amino acid sequence shown as SEQ ID NO.2, and CDR3 has the amino acid sequence shown as SEQ ID NO. 3;

(2) CDR1 has the amino acid sequence shown as SEQ ID NO.7, CDR2 has the amino acid sequence shown as SEQ ID NO.9, and CDR3 has the amino acid sequence shown as SEQ ID NO. 3;

(3) CDR1 has the amino acid sequence shown as SEQ ID NO.7, CDR2 has the amino acid sequence shown as SEQ ID NO.8, and CDR3 has the amino acid sequence shown as SEQ ID NO. 3;

(4) CDR1 has the amino acid sequence shown as SEQ ID NO.7, CDR2 has the amino acid sequence shown as SEQ ID NO.8, and CDR3 has the amino acid sequence shown as SEQ ID NO. 10;

the CDRs of the light chain variable region are any one of the following:

(1) CDR1 has the amino acid sequence shown as SEQ ID NO.4, CDR2 has the amino acid sequence shown as SEQ ID NO.5, and CDR3 has the amino acid sequence shown as SEQ ID NO. 6;

(2) CDR1 has the amino acid sequence shown as SEQ ID NO.4, CDR2 has the amino acid sequence shown as SEQ ID NO.13, and CDR3 has the amino acid sequence shown as SEQ ID NO. 15;

(3) CDR1 has the amino acid sequence shown as SEQ ID NO.11, CDR2 has the amino acid sequence shown as SEQ ID NO.13, and CDR3 has the amino acid sequence shown as SEQ ID NO. 15;

(4) CDR1 has the amino acid sequence shown as SEQ ID NO.12, CDR2 has the amino acid sequence shown as SEQ ID NO.13, and CDR3 has the amino acid sequence shown as SEQ ID NO. 15;

(5) CDR1 has the amino acid sequence shown as SEQ ID NO.12, CDR2 has the amino acid sequence shown as SEQ ID NO.14, and CDR3 has the amino acid sequence shown as SEQ ID NO. 15;

(6) CDR1 has the amino acid sequence shown as SEQ ID NO.4, CDR2 has the amino acid sequence shown as SEQ ID NO.14, and CDR3 has the amino acid sequence shown as SEQ ID NO. 15;

Among the above antibodies, the following antibodies have higher TROP2 binding affinity, and the CDRs of the heavy chain variable region and the CDRs of the light chain variable region thereof are any one of the following:

(1) CDR1 of the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.1, CDR2 has an amino acid sequence shown as SEQ ID NO.2, and CDR3 has an amino acid sequence shown as SEQ ID NO. 3; CDR1 of the light chain variable region has an amino acid sequence shown as SEQ ID NO.4, CDR2 has an amino acid sequence shown as SEQ ID NO.5, and CDR3 has an amino acid sequence shown as SEQ ID NO. 6;

(2) CDR1 of the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.7, CDR2 has an amino acid sequence shown as SEQ ID NO.8, and CDR3 has an amino acid sequence shown as SEQ ID NO. 3; CDR1 of the light chain variable region has an amino acid sequence shown as SEQ ID NO.4, CDR2 has an amino acid sequence shown as SEQ ID NO.13, and CDR3 has an amino acid sequence shown as SEQ ID NO. 15;

(3) CDR1 of the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.7, CDR2 has an amino acid sequence shown as SEQ ID NO.8, and CDR3 has an amino acid sequence shown as SEQ ID NO. 3; CDR1 of the light chain variable region has an amino acid sequence shown as SEQ ID NO.12, CDR2 has an amino acid sequence shown as SEQ ID NO.13, and CDR3 has an amino acid sequence shown as SEQ ID NO. 15;

(4) CDR1 of the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.7, CDR2 has an amino acid sequence shown as SEQ ID NO.8, and CDR3 has an amino acid sequence shown as SEQ ID NO. 3; CDR1 of the light chain variable region has the amino acid sequence shown as SEQ ID NO.12, CDR2 has the amino acid sequence shown as SEQ ID NO.14, and CDR3 has the amino acid sequence shown as SEQ ID NO. 15.

Based on the CDR regions of the heavy and light chain variable regions described above, the present invention provides the sequences of the heavy and light chain variable regions of these TROP2 antibodies:

The heavy chain variable region has the amino acid sequence shown in any one of SEQ ID NO.16 and 18-20, and the light chain variable region has the amino acid sequence shown in any one of SEQ ID NO.17 and 21-25;

or the heavy chain variable region and the light chain variable region have at least one of the following compared with the aforementioned sequences: a) Binding to the same epitope; b) Amino acid sequence having greater than 70%, 80%, 85%, 90%, 97%, 98% or 99% sequence identity.

Further, antibodies with the following variable region sequences exhibited more excellent TROP2 binding affinity: the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.16, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 17;

or the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.19, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 21;

Or the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.19, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 23;

Or the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.19, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 24.

Alternatively, the Fc fragment of the heavy chain of a TROP2 antibody described above is the Fc fragment of a human or humanized antibody that is IgG1, igG2, igA, igE, igM, igG4 or IgD.

Preferably, the heavy chain of the TROP2 antibody has the amino acid sequence shown in any one of SEQ ID nos. 27, 29, and the light chain has the amino acid sequence shown in any one of SEQ ID nos. 28, 30; or the heavy chain and the light chain have at least one of the following compared with the sequences: a) Binding to the same epitope; b) Amino acid sequence having greater than 70%, 80%, 85%, 90%, 97%, 98% or 99% sequence identity.

The present invention provides TROP2 antibodies having the following heavy and light chain full length sequences: the heavy chain has an amino acid sequence shown as SEQ ID NO.27, and the light chain has an amino acid sequence shown as SEQ ID NO. 28; or the heavy chain has the amino acid sequence shown in SEQ ID NO.29, and the light chain has the amino acid sequence shown in SEQ ID NO. 30.

The human anti-human TROP2 antibody provided by the invention binds to human TROP2 with an affinity of 4.35nM-7.5 nM. The antibody binds to a cell expressing TROP2, which can be a human epithelial malignancy (tumor such as gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, esophageal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, intrauterine mucosal serous papillary carcinoma, etc.).

The invention also provides single chain antibodies, fab antibodies, minibodies, chimeric antibodies, whole antibodies immunoglobulins IgG1, igG2, igA, igE, igM, igG4 or IgD, bispecific antibodies, multispecific antibodies comprising a TROP2 antibody as described above.

Further, the invention provides a bispecific antibody that binds TROP2 and CD3 comprising: a first domain that binds trophoblast cell surface antigen 2 and a second domain that binds T cell surface antigen CD3, wherein the first domain comprises a heavy chain variable region and a light chain variable region that are the heavy chain variable region and the light chain variable region of a TROP2 antibody as described above.

Preferably, the first domain of the bispecific antibody is 2 intact light chain-heavy chain pairs linked by disulfide bonds, the amino acid sequences of the heavy and light chains of which are the amino acid sequences of the heavy and light chains of the TROP2 antibody described above.

For the second domain that binds CD3 antigen, the heavy chain variable region preferably has the amino acid sequence shown in SEQ ID No.31, and the light chain variable region preferably has the amino acid sequence shown in SEQ ID No. 32;

Preferably, the light chain variable region and the heavy chain variable region of the second domain are linked by a linker peptide to form a single chain antibody having the amino acid sequence shown in SEQ ID NO. 33.

The bispecific antibodies of the invention are preferably designed to have the following structure: the first domain comprises 2 complete light chain-heavy chain pairs and the second domain comprises 2 single chain antibodies, symmetrically linked by any one of the following:

(1) The C-terminal of the 2 single-chain antibodies of the second domain are respectively connected with the N-terminal of the 2 heavy chains of the first domain through connecting peptides;

(2) The N ends of the 2 single-chain antibodies of the second structural domain are respectively connected with the C ends of the 2 heavy chains of the first structural domain through connecting peptides;

The invention discovers that the bispecific antibody with the symmetrical structure can better retain the specific antigen binding capacity of the primary antibodies of the first structural domain and the second structural domain compared with the bispecific antibodies with other structures aiming at the sequences of the first structural domain and the second structural domain, has excellent biological functions of binding TROP2 and CD3, and has obvious advantages in the aspects of production process, medicinal performance and the like. The invention develops the bispecific antibody which combines TROP2 and CD3 and has the molecular structure of the antibody, and the bispecific antibody has specific targeting effect and can efficiently excite immune response with guidance and kill tumor cells.

Preferably, the amino acid sequence of the connecting peptide for linking the first domain and the second domain is (GGGGX) n, wherein X is Gly or Ser and n is a natural number from 1 to 4.

As a preferred embodiment of the present invention, the amino acid sequence of the connecting peptide is shown in SEQ ID NO. 34.

As an example of a bispecific antibody having the above structure, the present invention constructs a bispecific antibody that binds both TROP2 and CD3 on the basis of the above TROP2 antibody and CD3 antibody, the structure and sequence of which are as follows:

The heavy chain of the first domain and the second domain are connected through a connecting peptide and have the amino acid sequence shown in SEQ ID NO.35 or 36, and the light chain has the amino acid sequence shown in SEQ ID NO.28 or 30.

Preferably, the heavy chain of the first domain has an amino acid sequence shown in SEQ ID NO.35 after being connected with the second domain through a connecting peptide, the light chain has an amino acid sequence shown in SEQ ID NO.28, or the heavy chain of the first domain has an amino acid sequence shown in SEQ ID NO.36 after being connected with the second domain through a connecting peptide, and the light chain has an amino acid sequence shown in SEQ ID NO. 30.

The sequences shown in SEQ ID NOS.1-36, which are disclosed and claimed above, include "conservative sequence modifications", i.e., nucleotide and amino acid sequence modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody containing the amino acid sequence. The conservative sequence modifications include nucleotide or amino acid substitutions, additions or deletions. Modifications may be introduced into SEQ ID NOS.1-36 by standard techniques in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, and conservative amino acid substitutions include amino acid residues being replaced with amino acid residues having similar side chains or with other amino acid residues. In the art, families of amino acid residues with similar side chains have been defined. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace an optional amino acid residue in a human anti-TROP 2 antibody with another amino acid residue from the same side chain family.

Thus, the above disclosed antibodies having the amino acid sequences and/or antibodies comprising the above disclosed amino acid sequences, including antibodies substantially encoded by or comprising similar sequences modified by a conserved sequence, are all considered within the scope of the present invention.

The invention also provides nucleic acid molecules encoding the TROP2 antibodies and nucleic acid molecules encoding the bispecific antibodies that bind TROP2 and CD 3.

In view of the degeneracy of codons, genes encoding the antibodies of the invention may be modified in their coding regions, without altering the amino acid sequence, to obtain genes encoding the same antibodies. Those skilled in the art can artificially synthesize engineered genes to increase the expression efficiency of antibodies according to the codon preference of the host expressing the antibodies.

The invention also provides biological materials comprising the nucleic acid molecules, including recombinant DNA, expression cassettes, vectors, host cells, engineered bacteria, or cell lines.

Wherein the vector includes, but is not limited to, cloning vectors, expression vectors, and can be plasmid vectors, viral vectors, transposons, and the like.

The host cell or cell line may be a cell or cell line of microbial or animal origin.

The invention also provides a method of preparing the TROP2 antibody or the bispecific antibody that binds TROP2 and CD3 comprising: introducing a nucleic acid encoding said antibody into a host cell to obtain a host cell stably expressing said bispecific antibody; culturing host cells, and separating and purifying to obtain the antibody.

In preparing the TROP2 antibody or the bispecific antibody that binds TROP2 and CD3, one skilled in the art can select host cells, expression vectors, methods of introducing expression vectors into host cells, and methods of isolating and purifying antibodies as desired, which are conventional in the art.

Based on the functions of the TROP2 antibody and the bispecific antibody binding to TROP2 and CD3 provided by the invention, the invention provides any one of the following applications of the TROP2 antibody, the bispecific antibody binding to TROP2 and CD3, the nucleic acid molecule encoding the antibody, and the biological material containing the nucleic acid molecule:

(1) Use in the manufacture of a medicament for the diagnosis, prevention or treatment of a disease associated with TROP2 expression;

(2) The use in the manufacture of a medicament for the diagnosis, prevention or treatment of a disease targeted at TROP 2;

(3) Use in the manufacture of a medicament for killing a cell expressing TROP 2;

(4) The application in preparing TROP2 and/or CD3 detection reagent;

(5) Use in the preparation of a relevant agent suitable for CAR-T therapy;

(6) Use in the preparation of immunotoxins or labeled antibodies.

The above-mentioned diseases associated with TROP2 expression are preferably tumors that highly/overexpress TROP2, in particular epithelial malignancies expressing TROP2 (including but not limited to tumors of gastric, cervical, breast, lung, prostate, colon, esophageal, pancreatic, head and neck, ovarian, intrauterine mucosal serous papillary carcinoma, etc.).

Preferably, the drug is an anti-tumor drug.

The labeled antibody described above may be a radioisotope labeled antibody.

The bispecific antibodies provided by the invention are useful per se in therapy and diagnosis. Antibodies can be labeled, cross-linked or conjugated and expressed in fusion with other protein or polypeptide molecules to form complexes (e.g., cytotoxic substances, radioactive toxins, and/or chemical molecules, etc.) for diagnostic and therapeutic use.

The present invention provides a multispecific antibody, fusion protein, immunotoxin, drug or detection reagent comprising the TROP2 antibody or the bispecific antibody that binds TROP2 and CD 3.

The immunotoxins described above comprise a TROP2 antibody or bispecific antibody linked to a cytotoxic agent in various forms.

The various connection forms are labeled, in vitro crosslinked or molecule coupled. Such cytotoxic agents include chemical molecules, radioisotopes, polypeptides, toxins, and other substances that have killing or cell death inducing properties on cells.

The fusion protein comprises a complex of the TROP2 antibody or the bispecific antibody and other protein or polypeptide molecules with certain functions.

Specifically, the fusion protein can be recombinant expression vector constructed by connecting antibody genes with immunotoxin or cytokine genes, and recombinant fusion protein molecules can be obtained by mammalian cells or other expression systems.

The medicaments and test agents described above may also contain other active ingredients or adjuvants (e.g., components of antibodies and pharmacologically acceptable delivery molecules or solutions: wherein the therapeutic components are sterile and lyophilized) as permitted in the pharmaceutical and test agent arts.

The anti-TROP 2 antibody and the bispecific antibody thereof provided by the invention can inhibit one or more biological activities induced by TROP 2. These antibodies act by internalizing the complex upon binding to TROP2, thereby depleting the cell surface of TROP 2. All interfering functions possessed by TROP2 antagonists are equally considered to be objects of the present invention.

The invention has the beneficial effects that:

the invention screens out the specific antibody of the anti-TROP 2 from the single chain antibody library of the natural whole human sequence by the genetic engineering and phage surface display library technology, the TROP2 antibody provided by the invention has good capability of combining with TROP2, and the antibody antibodies can be combined with the TROP2 specificity of MDA-MB-231, MDA-MB-468, MCF7 and other cell surfaces by ELISA detection and flow cytometry detection, thus indicating that the target specificity is good, the affinity of the antibody combined with human TROP2 is 4.35nM-7.5nM, and the antibody has good therapeutic application prospect.

On the basis, the invention utilizes genetic engineering and antibody engineering methods to construct the bispecific antibody which contains single chain antibody and complete monoclonal antibody structure and combines TROP2 and CD3, the bispecific antibody fusion protein retains the complete monoclonal antibody structure and has a highly stable symmetrical structure, better retains the biological functions of the anti-CD 3 single chain antibody and the anti-TROP 2 monoclonal antibody, realizes the biological functions of one bispecific antibody molecule and simultaneously has excellent anti-TROP 2 and anti-CD 3 monoclonal antibodies, can build a bridge between tumor cells and immune effector cells, effectively activates immune effector cells and guidance immune response, obviously enhances the efficacy of killing tumor cells by immune cells, simultaneously reduces the ADCC effect to the greatest extent, and has higher safety. In addition, the bispecific antibody provided by the invention has the characteristic of complete symmetry in structure, and can not generate protein isomers with other structures when in host expression, thereby greatly reducing the difficulty of extraction and purification processes, having the advantages of simple preparation and high yield, and having wide application prospects in the immunotherapy of tumors.

The invention provides human TROP2 specific antibody candidate molecules for developing anti-tumor antibody medicaments aiming at TROP2 targets and developing, preventing and treating CAR-T reagents. The bispecific antibody can combine immune cells and tumor cells simultaneously, guide T immune response, specifically and effectively kill the tumor cells, and can be developed into an antibody medicament for epithelial malignant tumor.

Drawings

FIG. 1 is a schematic diagram of the protein structure and TCR structure of TROP2 in the background art of the invention, wherein A is the structure of TROP2 antigen, and source ：Goldenberg DM,Stein R,Sharkey RM.The emergence of trophoblast cell-surface antigen 2(TROP-2)as a novel cancer target.Oncotarget.2018Jun 22;9(48):28989-29006.doi:10.18632/oncotarget.25615.PMID:29989029;PMCID:PMC6034748;B is the structure of the T Cell Receptor (TCR) complex and the composition of CD3 therein. The source is as follows: https:// www.researchgate.net/publication/299549376.

FIG. 2 is a flow cytometer (FACS) analysis of the binding of TROP2 to the lead clone 1F7 of example 2 of the present invention, wherein A is a negative control; b: a positive control; c: candidate clone IF7.

FIG. 3 is a graph showing the plasma resonance (SPR) technique of example 3 of the present invention for analyzing the affinity of 1F7 and its affinity matured mutant F7A for human and cynomolgus TROP2, wherein the ordinate indicates the binding Response Unit (RU).

FIG. 4 is a schematic structural diagram of an anti-TROP 2 XCD 3 bispecific antibody constructed based on the FIST platform in example 4 of the present invention, wherein A is a diabody constructed by N-terminal fusion (nFIST): the anti-CD 3 single-chain antibody is fused to the N end of the anti-TROP 2 antibody VH; b, a double antibody constructed by C-terminal fusion (cFIST): the anti-CD 3 single chain antibody is fused to the C-terminus of the anti-TROP 2 antibody Fc.

FIG. 5 shows the affinity of the single chain antibody K3 for CD3 in the SPR assay of example 4 of the present invention.

FIG. 6 is a SDS-PAGE electrophoresis of bispecific antibodies F7AK3 and 1F7K3 in example 5 of the present invention, wherein A and C are reducing SDS-PAGE electrophoresis; b and D are non-reducing SDS-PAGE electrophoresis; a and B are the results of SDS-PAGE of 1F7K3 bispecific antibodies; c and D are the results of SDS-PAGE electrophoresis of F7AK3 bispecific antibodies; lane M represents a protein molecular weight standard and lane 1 is a protein of interest.

FIG. 7 is a graph showing the HPLC-SEC purity analysis peak profile of the purified bispecific antibodies F7AK3 and K3F7A in example 5 of the present invention, wherein A is the bispecific antibody F7AK3; b is a bispecific antibody 1F7 K3.

FIG. 8 shows the flow cytometer analysis of F7AK3 binding to TROP2 and F7AK3 binding to T cells in example 6 of the present invention, wherein A is the flow cytometry analysis of F7AK3 binding to TROP2 cell line; B-G: gradient analysis of F7AK3 binding capacity to TROP2 cell lines;

FIG. 9 shows the bridging effect of F7AK3 on TROP 2-positive cells and T cells according to example 6 of the present invention.

FIG. 10 shows the detection of the killing ability of human breast cancer cells (MCF, MDA-MB-231, MDA-MB-468, HCC 1395) by T cells mediated by binding of F7AK3 diabodies to TROP2 antigen in example 7 of the present invention, wherein A, B, C, D is MCF, MDA-MB-231, MDA-MB-468, HCC1395 cells, respectively.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Example 1 screening of anti-human TROP2 antibodies from a Natural human antibody phage surface display library

The antibody library technique is a technique of cloning all antibody variable region genes of a certain animal (including human) into plasmids or phages, and the latter express antibody fragments on the surface of phage particles or in the periplasm and cytoplasm of E.coli after infection with E.coli. And then screening clones carrying specific antibody genes from the antibody library by using target antigens, thereby obtaining corresponding specific antibodies. Antibodies required in various basic studies and clinical developments have been selected from antibody libraries, such as tumor-associated membrane protein antigens, autoantigens associated with autoimmune diseases, antibodies against viral antigens of viral diseases. These show great potential for application of antibody library technology in basic research and antibody drug development. In particular, the fully human monoclonal antibody is obtained from a human antibody library, thereby overcoming the obstacle that the human monoclonal antibody is difficult to obtain by the mouse hybridoma technology; because of the high degree of conservation of human antibody sequences, these antibodies are far less immunogenic to the human autoimmune system than antibodies derived from animals and are therefore safer.

The non-immunized natural antibody repertoire is derived from healthy human antibody genes, and these humans are called donors. Due to the use of fully natural human antibody sequences, the resulting antibodies have very low immunogenicity to the human immune system. After total RNA was extracted from Peripheral Blood Mononuclear Cells (PBMC) of the donor using RNA extraction kits (e.g., QIAGEN RNEASY MINI KIT, catalog No. 74104), equal amounts of total RNA were pooled from each RNA sample, synthesis of light and heavy chain gene cDNAs was performed using reverse transcription kits (e.g., thermoFisher Scientific cDNA synthesis kit (SuperScript ^TM IV REVERSE TRANSCRIPTASE)), using these antibody cDNAs as templates, PCR amplification was performed in the first round with each subunit-specific upstream primer and constant region primer, and all member genes of each subunit were obtained, in the second round of PCR, the heavy chain variable region (VH) 3' -end primer set with a bridge and the light chain variable region (VL) 5' -end primer set with a bridge were engaged with the respective other end primer (containing specific restriction sites) so that the obtained VH and VL genes were head-linked to form a single chain antibody (scFv) gene, and the single chain gene (scFv) was constructed using these antibody genes at 3' -end primers and 3' -end primers to 3' -end primers of the scFv gene library (3482) in the second round of PCR, and the single chain gene library was constructed as a large clone library (32. Mu.50).

Biopanning refers to the process of screening a pool of antibodies for specific monoclonal antibodies with a specific target. To obtain human antibodies specific for the human TROP2 antigen, panning was performed using a liquid phase screening method. The general steps of the liquid phase panning method are: commercial TROP2 antigen (TROP 2-Fc fusion protein Acrobiosystems, cat. No. TR 2-H5253) was first modified with biotin to crosslink 3-5 biotin molecules per molecule. A suitable amount of biotin-labeled TROP2 antigen is bound to streptavidin-conjugated magnetic beads (e.g., dynabeads ^TM M-280Streptavidin,ThermoFisher Scientific, cat. No. 11205D). A pool of human antibodies was thawed and contained 100 million phage particles expressing different antibodies. Both the beads and antibody libraries were blocked with 4% nonfat milk powder solution (4% MPBS) to eliminate non-specific sites of action. Since there may be streptavidin on the beads that is not bound by avidin, and since the TROP2 antigen molecules are fusion proteins made up of human Fc, it is necessary to add sufficient amounts (50-100 fold excess over the amount of target protein used) of streptavidin and human antibody Fc simultaneously to the thawed antibody pool to remove antibody clones that bind them. The blocked antibody library solution was added to a 1.5ml Eppendorf tube along with magnetic beads (total volume <1 ml) and incubated with spin mixing at room temperature for 2 hours to allow binding of TROP2 to specific phage antibodies. After the incubation was completed, the Eppendorf tube was placed on a magnetic rack and allowed to stand for 1 minute, and the magnetic beads and the solution were separated. The solution part is removed completely as much as possible by a pipette, a new tip (tip) is replaced, 1ml of washing solution PBST (Tween 20 with the final concentration of 0.05% is added into Phosphate Buffer Solution (PBS)) is added into the Eppendorf tube, the magnetic force frame is far away, the magnetic beads are suspended lightly by the pipette, and the magnetic beads and the solution are separated by being placed on the magnetic force frame again. The solution was removed and this was repeated three times. Then the washing solution was changed to PBS and the beads were washed 3 times. After washing, the nonspecific and low affinity phage antibodies are mostly removed, and the specific phage antibodies remain on the beads. The first round of panning was completed by adding eluent (10mM Glycine,pH2.0) to the beads, resuspending the beads, standing at room temperature for 10 minutes, separating the beads and the solution with a magnetic rack, pipetting the solution into a clean Eppendorf tube, adding 1/10 1m Tris solution (ph 8.0) to neutralize the solution, which is the eluent containing thousands of phage antibodies with different binding TROP2 antigen affinities.

To further harvest specific antibody clones with higher affinity, more rounds of panning were required. To this end, the log phase E.coli (e.g.TG 1 strain) which is infected with M13 phage was infected with phage antibody solution eluted by the first round of panning to obtain an infection solution. A series of 10-fold gradient dilutions (typically to parts per million of stock solution and coating with the last three gradients) were performed on small amounts of the infection solution to determine the titer (titer) of the first round of output eluate (output), also referred to as the first round of maximum diversity, titer, typically with output titers below 10E6 cfu after the first round of panning. Coating all other infection liquid on a bacterial culture plate containing corresponding antibiotics for overnight culture to obtain bacterial colonies; the colony layer was scraped and resuspended in liquid medium, and sufficient amount of the resuspension containing the first round of output diversity was taken into shake flasks containing sufficient amount of liquid medium (2 YT-CG,2YT medium with final concentrations of Carbenicillin and glucose of 100. Mu.g/ml and 2%, respectively) and the resuspension was diluted below 0.1OD600 and started to culture until the logarithmic phase, OD ₆₀₀, reached around 0.5. To allow the antibodies obtained in the first round of panning to reappear to the phage particle surface, 10ml of bacterial liquid was taken, helper phage M13K07 was added to give a multiplicity of infection of 20:1, and the mixture was allowed to stand at 37℃for 30 minutes (this stage is called phage rescue). Centrifugation was performed, and cells were resuspended in 50ml of an expression medium (2 YT-AK, carbenicillin and KANAMYCIN were added to the 2YT medium, and the final concentrations were 100. Mu.g/ml and 30. Mu.g/ml, respectively) and incubated at 30℃for 200 revolutions/minute overnight. The culture supernatant was harvested by centrifugation the next day, 1/5 volume of PEG8000/NaCl (PEG-8000 20%, naCl 2.5M) was added, thoroughly mixed, and incubated on ice for 1 hour. High speed centrifugation (11500 Xg) for 30 minutes to harvest phage antibody particles. The pellet was resuspended in 1ml PBS and centrifuged again at high speed to remove bacterial debris. The supernatant is the amplification solution after the first round of panning, and each antibody clone contained in the supernatant is amplified by more than ten thousand times. This amplification solution was used for the second round of panning experiments. The second round of panning was performed exactly the same way as the first round except that in PBST/PBS, the procedure was increased to 6 times each (6/6). In the third wheel, the number of washes can be further increased to 10/10. Multiple rounds of panning will generally be effective in enriching for specific clones, although diversity is significantly reduced, they are all relatively high in affinity, facilitating subsequent monoclonal screening.

In order to obtain specific monoclonal antibodies, a monoclonal phage enzyme-linked immunosorbent assay (Monophage ELISA) is performed. For this purpose, single colonies well isolated in the second and/or third rounds of gradient dilution were individually inoculated into 96-well plates containing 2YT-AG (93 colonies were inoculated per plate leaving three wells as negative controls) and incubated overnight, which is the master (MASTER PLATE). Bacterial liquid from each well in the master plate is inoculated into a new culture plate to grow to a logarithmic phase, and phage rescue is carried out to ensure that antibodies of each clone are expressed on the surface of phage. TROP2 antigen (1. Mu.g/ml) was coated on a conventional 96-well enzyme-linked plate. Each separately expressed monoclonal phage antibody bacterial solution is added to a TROP2 plate respectively, then a proper secondary antibody (mouse anti-M13 monoclonal antibody) and a horseradish peroxidase (HRP) coupled tertiary antibody (rabbit anti-mouse polyclonal antibody) are connected, an HRP substrate is added for color development, and the absorbance value (450 nM) is read. The method for judging TROP2 positive clone comprises the following steps: the signal is 3 times higher than that of the negative control hole. Clones corresponding to multiple wells showed positive for TROP2 antigen, collectively referred to as hit, by analysis.

These hit bacteria were inoculated to 3ml of 2YT-CG from the corresponding wells of the master plate, and incubated at 37℃for 200 rpm overnight. The next day phagemid DNA was extracted and the sequence of the single-chain antibody region containing each hit was determined with specific primers. Coding region DNA sequences were translated into amino acid sequences and subjected to multiple sequence comparisons (CLUSTALW, website linking https:// www.genome.jp/tools-bin/CLUSTALW) to determine clone specificity. These hits were analyzed to be in sequence in two different clones, one of which was designated 1F7, whereby the fully human antibody variable region sequence was obtained against the human TROP2 antigen. The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of 1F7 are shown as SEQ ID NO.1-3 respectively, the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO.4-6 respectively, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.16, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17. The amino acid sequence of the single-chain antibody consisting of the light chain variable region and the heavy chain variable region of 1F7 is shown in SEQ ID NO. 26.

Example 2 antibody binding and functional validation

To verify whether the obtained TROP2 antibody clone bound to the purified TROP2 antigen and the TROP2 expressed on the cell membrane surface, the gene of the 1F7 single chain antibody was cloned into eukaryotic expression vector pFH (manufactured by beneficial kosmo corporation) to obtain plasmid pFH-1F7. In this vector, the scFv gene and the Fc gene of human IgG4 are fused to express a protein in the form of scFv-Fc, which can be affinity purified using Potein-A or detected using (HRP or fluorescein) -labeled anti-human Fc antibodies.

After obtaining the scFv-Fc protein of 1F7, binding of 1F7 to CHO cell line expressing TROP2 (manufactured by beneficial cosite) was examined by flow cytometry, confirming that 1F7 specifically bound to TROP2 antigen on the cell membrane surface (see fig. 2). The percentage of corners (percent positive) in the graph represent the strength of binding of the antibody to the cell surface antigen. The positive percentage of clone 1F7 was 89.33%, thus verifying the binding capacity of the antibody to the human cell surface antigen TROP 2.

Example 3 affinity maturation of antibodies

In vitro affinity maturation is a rapid directed molecular evolution operation: mutations were introduced at the DNA level and screening was performed at the protein binding level. For the 1F7 antibody, in order to avoid introducing mutations that may enhance immunogenicity or significantly alter the structure of the antibody, the mutation sites are limited to the sequences of the CDR regions, and saturation mutations are performed at each position, and each mutation is expressed separately and detected separately.

The affinity maturation process is: first, the light and heavy chain variable region genes of 1F7 were cloned into pUFL vectors (pUFL is a plasmid capable of expressing antibody Fab in E.coli, manufactured by Yikeshite Co., ltd.) to give the Fab form of 1F 7. A complete set of CDR region single-point random mutation primers are designed, PCR mutation is carried out on each CDR position by a point mutation kit (QuikChange Lightning Multi Site-Directed Mutagenesis Kit, agilent catalog number 210515) respectively, BL21 (DE 3) competent bacteria are transformed respectively, and monoclonal colony plates are prepared respectively, and the mutants are collectively called a single-point mutation library. The Fab vector of parent 1F7 was also transformed. >30 colony preparation DNAs were picked and sequence analysis was performed on the mutant regions to evaluate mutation efficiency. 92 colonies were picked per mutation at each CDR position when the mutation efficiency was appropriate (greater than 80%) into 96-well plates containing the corresponding medium (100. Mu.g/ml Carbenicillin +0.1% glucose in 2 YT), and 3 parental colonies were picked, leaving three wells as blank. Plates were incubated at 37℃for 6 hours at 300 rpm, IPTG was added to a final concentration of 1mM, and the plates were transferred to 30℃at 300 rpm overnight, and Fab fragments were expressed and secreted into the medium.

Through pre-experiments, the enzyme-linked reaction conditions were optimized such that the binding a450 absorption value of the 1F7 parent antibody Fab to antigen TROP2 was slightly higher than about 3-fold of the background signal. TROP2 antigen was coated on the ELISA plate at 0.25. Mu.g/ml a day in advance, secretory mutant Fab was added in wells the next day, HRP-conjugated anti-human Fab was used as secondary antibody, substrate developed, A450 was read, and clones corresponding to wells with a signal 1.5 times higher than the highest value of the parental wells were obtained, and these clones were called hit. All hits from the light variable region were collected, re-induced and enzyme-linked experiments were performed to verify that they did have higher affinity than the parent antibody. The sequence analysis of the mutation region of the repeatedly positive cloned plasmid is performed to obtain the mutation information of the CDR amino acids, and the process is called primary screening (PRIMARY SCREENING). All mutation information of the CDR regions that is beneficial for affinity improvement is thus obtained: the mutation sites provided for affinity in the heavy chain variable region CDRs are shown in table 1, and the mutation sites provided for affinity in the light chain variable region CDRs are shown in table 2.

TABLE 1F7 mutations in the heavy chain variable region CDRs which are beneficial for affinity enhancement

CDR region location	Parent amino acids	Advantageously mutated amino acids
			H31	S	N
H53	N	R，K，G
			H99	G	H，A
H100	D	E

TABLE 2 mutations in the CDRs of the light chain variable region of 1F7 that are beneficial for affinity enhancement

CDR region location	Parent amino acids	Advantageously mutated amino acids
			L28	G	N,E
L30	S	R,K
			L50	A	R
L52	S	G,H,R
			L53	S	K

After all individual mutation information of the whole CDR region beneficial to affinity improvement is obtained from the primary library by enzyme-linked experimental screening, new multi-point mutation primers are redesigned to contain the main beneficial mutations, and a mutation library is constructed again with a point mutation kit, which library is called a combinatorial library. To screen as many combinations of mutations as possible in the light and heavy chain variable regions, combinatorial libraries of light and heavy chain variable regions comprising a respective plurality of mutations are constructed and screened separately. The first 10 clones with highest affinity are screened from a light chain variable region group library of a parent heavy chain variable region, the light chain variable region amino acid sequences of the first 10 clones comprise 5 mutant sequences, the sequences are shown as SEQ ID NO.21-25, CDR1 of the light chain variable region has any one of the amino acid sequences shown as SEQ ID NO.4, 11 and 12, CDR2 has any one of the amino acid sequences shown as SEQ ID NO.2, 13 and 14, and CDR3 has any one of the amino acid sequences shown as SEQ ID NO.3 and 15.

The amino acid sequences of 10 clones with highest affinity are obtained by screening in a heavy chain variable region group library of a parent light chain variable region, the amino acid sequences of the heavy chain variable regions comprise 3 mutation sequences, which are respectively shown as SEQ ID NO.18-20, CDR1 of the heavy chain variable region has any one of the amino acid sequences shown as SEQ ID NO.1 and 7, CDR2 has any one of the amino acid sequences shown as SEQ ID NO.2, 8 and 9, and CDR3 has any one of the amino acid sequences shown as SEQ ID NO.3 and 10.

Meanwhile, mixing the positive all heavy chain variable region mixed clone obtained by the screening and all positive light chain variable region clone, respectively preparing DNA mixed plasmids of the positive all heavy chain variable region mixed clone and the positive light chain variable region clone, and assembling the DNA mixed plasmids into a final Fab antibody combined library in an enzyme digestion and linked recombination mode. Screening the library, picking the first 10 positions with the highest signals, analyzing the DNA sequence of the library to obtain 3 different Fab clones which have the same heavy chain variable region and the amino acid sequence of which is shown as SEQ ID NO. 19; the amino acid sequences of the light chain variable regions are shown in SEQ ID NO. 21-24.

The TROP2 antibodies obtained by screening are tested for the TROP2 binding capacity with different cell lines by using a flow cytometer (FACS), and the results show that the TROP2 antibodies are relative to the parent antibody 1F 7; the affinity-improved polyclonal antibodies (F7A, F7B, F7C) exhibited significantly improved apparent affinity in flow cytometric analysis. ELISA detection showed that the intrinsic affinity of parent antibody 1F7 was about 7.5nM, while the intrinsic affinity of mutant F7A was 4.35nM.

To examine whether the affinity of the obtained mutants was improved, one of the mutants F7A and the 1F7 parent antibody in IgG form was prepared, expressed and purified, respectively. 1F7 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 3; the amino acid sequence of CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is shown as SEQ ID NO.5, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 6; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.16, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17; the full-length sequence of the light chain is shown as SEQ ID NO.30, and the full-length sequence of the heavy chain is shown as SEQ ID NO. 29. Wherein the amino acid sequence of CDR1 of the heavy chain variable region of F7A is shown as SEQ ID NO.7, the amino acid sequence of CDR2 is shown as SEQ ID NO.8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3; the amino acid sequence of CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is shown as SEQ ID NO.13, the amino acid sequence of CDR3 is shown as SEQ ID NO.15, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.19, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 21; the full-length sequence of the light chain is shown as SEQ ID NO.28, and the full-length sequence of the heavy chain is shown as SEQ ID NO. 27. The expressed, purified antibody products were examined by Surface Plasmon Resonance (SPR) and their binding curves to the TROP2 proteins of humans and cynomolgus monkeys are shown in figure 3. The affinity assay data are shown in table 3. The results indicate that mutant F7A has a 1.74-fold improvement in binding to human TROP2 compared to 1F 7; the affinity to monkey TROP2 is improved by 3.18 times. The human and monkey crossover factor was reduced from 5.38 to 2.94 (table 3).

TABLE 3 SPR analysis of affinity of 1F7 and F7A for human and monkey TROP2

Antibodies to	Antigens	ka(1/Ms)	kd(1/s)	Affinity KD (M)
					1F7	Human TROP-2	1.18E+05	8.92E-04	7.57E-09
1F7	Rhesus TROP-2	2.31E+04	9.42E-04	4.07E-08
					F7A	Human TROP-2	1.92E+05	8.34E-04	4.35E-09
F7A	Rhesus TROP-2	1.28E+05	1.65E-03	1.28E-08

Example 4 TROP2×CD3 bispecific antibody design

In this example, bispecific antibodies were designed using tumor cell surface antigen TROP2 and immune cell surface antigen CD3 as targets.

Combining protein structure design software and a large number of manual experimental screens, the invention screens bispecific antibody structures with symmetrical structures comprising single chain antibody units and monoclonal antibody units from a plurality of bispecific antibody structures combining TROP2 and CD 3. The present invention refers to this technical platform as FIST (fusion of IGG AND SCFV technology). For example, an anti-TROP 2 monoclonal antibody unit may be an IgG antibody comprising 2 complete light chain-heavy chain pairs (i.e., comprising complete Fab and Fc domains, with disulfide linkages between the heavy and light chains), and anti-CD 3 may be a single chain antibody unit comprising 2 single chain antibodies (ScFv). The single-chain antibody and the monoclonal antibody are connected by adopting a connecting peptide, and the following connection mode can be designed for the connection mode of the single-chain antibody and the monoclonal antibody, so that the bispecific antibody with symmetrical structure can be obtained, and the structure schematic diagram is shown in figure 4.

The C-terminus of the single-chain antibody was connected to the N-terminus of the heavy chain variable region (VH) of the monoclonal antibody to give nFIST (A in FIG. 4). Single chain antibodies can also be fused to the C-terminus of IgG-Fc via a linker peptide to give cFIST (FIG. 4B). The sequence of the variable region of the anti-CD 3 single chain antibody UCHT1 is named K3 (SEQ ID NO. 33) from literature (Beverley,P.C.&Callard,R.E.Distinctive functional characteristics ofhuman"T"lymphocytes defined by Erosetting or a monoclonal anti-T cell antibody(1981)Eur.J.Immunol.11,329-334) and is humanised (Shalaby et.al.,Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene.(1992)J Exp Med.Jan 1;175(1):217-25)., and the single chain antibody constructed by the anti-CD 3 single chain antibody UCHT1 comprises two cysteines (Cys) respectively inserted into a heavy chain variable region and a light chain variable region, a pair of inter-chain disulfide bonds are formed after folding, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.31, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 32. . The binding force of single-chain antibody form K3 to human CD3 was detected using surface plasmon resonance, and the results are shown in FIG. 5.

EXAMPLE 5 preparation of TROP2×CD3 bispecific antibodies

According to the cFIST form of the bispecific antibody coding gene designed in example 4 and cloned into the expression vector pG4HK, 2 bispecific antibodies F7AK3 and 1F7K3 are obtained, the heavy chain amino acid sequences of which are shown in SEQ ID NO.35, 36, respectively; the light chain amino acid sequences are shown as SEQ ID NO.28 and SEQ ID NO. 30 respectively. And respectively and stably transfecting expression plasmids corresponding to 1F7K3 and F7AK3 into CHO-K1, and expressing to prepare the double antibody protein.

To obtain bispecific antibodies of high purity, purification was performed as follows. (1) pretreatment of feed liquid: the supernatant of the fermentation culture was centrifuged at 2000rpm for 10min and then filtered with a 0.22. Mu.M filter. (2) affinity chromatography: the antibodies in the pretreated broth were captured by MabSelect SuRe affinity chromatography column (commercially available from GE company under the trade designation 18-5438-02), and after having equilibrated the column sufficiently with equilibration buffer (10mM PB,0.1M NaCl,pH7.0), the column was passed through affinity chromatography column and eluted with elution buffer (0.1M citric acid, pH 3.0). (3) cation exchange chromatography: samples prepared by affinity chromatography were further purified by molecular sieve exchange chromatography, buffer (50mM PBS,0.2Man Na ₂SO₄, pH 6.7).

1F7K3 and F7AK3 were subjected to SDS-PAGE and HPLC-SEC detection, the results of SDS-PAGE are shown in FIG. 6, and the results of HPLC-SEC detection are shown in FIG. 7. The detection result shows that the bispecific antibodies 1F7K3 and F7AK3 are successfully prepared through expression and purification, and the monomer purity of the bispecific antibody after purification is more than 95 percent.

Example 6 flow assay for detection of binding Activity of bispecific antibody F7AK3 to TROP2 cells and T cells

To verify the binding of F7AK3 to TROP2 cell lines and T cells, bispecific antibodies of F7A and single chain antibody K3 were constructed in cFIST format.

Breast cancer cell lines MCF, MDA-MB-231, MDA-MB-468, HCC1395, CD3 ⁺ T cells were resuspended in PBS at 1×10 ⁶ cells/reaction tube, the cells were rinsed once with 1ml binding buffer (PBS containing 0.5% w/v bsa+2mM EDTA), centrifuged (350×g,4 ℃ for 5 min) and then resuspended with 200 μl binding buffer. Bispecific antibody and F7AK3 were added to 5. Mu.g/ml and incubated on ice for 45min. Cells were resuspended in 100 μl binding buffer after rinsing the cells once as above. Mu.l of a fluorescent-labeled secondary antibody (PE anti-human IgG Fc Antibody, biolegend, 409304) was added to the sample tube. Cells were rinsed once as above. After resuspension of the cells with 200 μl PBS, detection was performed on-machine (Beckman). The flow chart for each cell line is shown in FIG. 8A. Binding gradient analysis is shown in FIGS. 8B-E, wherein binding to CD3 of human T cells is shown in FIG. 8F. EC50 analysis data are shown in table 4 and G of fig. 8. The results show that F7AK3 is capable of concentration-dependent binding to TNBC cell lines of different TROP2 expression levels, while also being capable of efficiently binding to CD3 of human T cells, with a 2-to 10-fold difference in EC50 (Table 4). Meanwhile, as shown in FIG. 9, F7AK3 can crosslink T cells and TNBC cancer cells (e.g., MDA-MB-468) to form a cell cluster (synapse).

TABLE 4 flow cytometry to detect F7AK3 binding to TROP2 ⁺ cell line and CD3 ⁺ T cells

	MCF	MDA-MB-231	MDA-MB-468	HCC1395	CD3 ⁺ T cells
						EC50(ng/ml)	204.8	89.06	66.77	53.24	568.3

Example 7 bispecific antibody mediated in vitro cell killing efficiency detection

In this example, MCF, MDA-MB-231, MDA-MB-468, HCC1395 and murine cell 4T1 were used as target cells, PBMC was used as immune effector cells, and the killing effect of bispecific antibody F7AK 3-mediated target cells was detected. The specific experimental steps are as follows:

1) Target cell preparation: target cells were cultured, counted after being blown up, centrifuged at 1000rpm for 5min, and washed once with PBS. After the target cells were washed by centrifugation, the density was adjusted to 0.2X10 ⁶/ml with GT-T551 medium, and 50. Mu.l per well was added, whereby 10000 cells were present per well.

2) PBMC preparation: PBMC were used as effector cells. PBMC frozen in a liquid nitrogen tank are taken out (reference cells are frozen and thawed), added into a 15ml centrifuge tube containing PBS or GT-T551 culture medium, centrifuged at 1000rpm for 5min, washed twice with PBS or GT-T551 culture medium, counted for cell number, activity and density, and the viable cell density is adjusted to 2X 10 ⁶/ml, 50 μl is added per well, and 100000 cells per well.

3) Antibody dilution: bispecific F7AK3 was diluted with GT-T551 medium to adjust the initial antibody concentration to 10nM. Sequentially mixing the components in 1: 5. 100 μl of diluted antibody was added to the prepared cells, mixed well, and the 96-well plate was returned to the incubator for 24 hours, and the killing effect was measured.

4) And (3) detection: dead living cells were differentiated by PMA staining, and then killing efficiency was calculated by cell counting.

5) And (3) data processing: the calculation formula of the target cell killing ratio is as follows:

target cell killing ratio (percent) =100× (target cell well read only-assay well read only)/assay well read only.

The antibody concentrations corresponding to the killing ratios of the target cells in all the detection wells were converted to log10, and curves were made with the abscissa and the killing ratio as the ordinate. The curves of the killing concentration-gradient of F7AK3 on sensitive cell strains MCF, MDA-MB-231, MDA-MB-468 and HCC1395 are shown in FIG. 10; the percent cell killing is shown in table 5. F7AK3 showed different killing ability for different TROP2 ⁺ cells.

TABLE 5 bispecific antibody mediated killing of target cells

	MCF	MDA-MB-231	MDA-MB-468	HCC1395
					Killing efficiency (%)	25	50	75	82

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Yikesitter (Beijing) pharmaceutical technology development Co., ltd

<120> An antibody binding to TROP2, bispecific antibodies targeting TROP2 and CD3, and preparation method and application thereof

<130> KHP211116562.5

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

Ser Tyr Gly Ile Asn

1 5

<210> 2

<211> 17

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 2

Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 3

<211> 11

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

Pro Asn Thr Gly Gly Asp Gly Ala Phe Asp Ile

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

Gln Gln Ala Asn Ser Phe Pro Ile Thr

1 5

<210> 7

<211> 5

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

Asn Tyr Gly Ile Asn

1 5

<210> 8

<211> 17

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

Trp Met Asn Pro Arg Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 9

<211> 17

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

Trp Met Asn Pro Gly Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 10

<211> 11

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

Pro Gly Thr Gly Gly Asp Gly Ala Phe Asp Ile

1 5 10

<210> 11

<211> 11

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

Arg Ala Ser Gln Glu Ile Lys Ser Trp Leu Ala

1 5 10

<210> 12

<211> 11

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

Arg Ala Ser Gln Gly Ile Lys Ser Trp Leu Ala

1 5 10

<210> 13

<211> 7

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

Arg Ala Arg Ser Leu Gln Ser

1 5

<210> 14

<211> 7

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 14

Arg Ala His Ser Leu Gln Ser

1 5

<210> 15

<211> 9

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 15

Gln Gln Ala Asn Ser Phe Pro Val Thr

1 5

<210> 16

<211> 120

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Thr Gly Arg Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Pro Asn Thr Gly Gly Asp Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 17

<211> 107

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 18

<211> 120

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Thr Gly Arg Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Gly Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Pro Asn Thr Gly Gly Asp Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 19

<211> 120

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Thr Gly Arg Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Arg Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Pro Asn Thr Gly Gly Asp Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 20

<211> 120

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Thr Gly Arg Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Arg Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Pro Gly Thr Gly Gly Asp Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 21

<211> 107

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 21

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Arg Ala Arg Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Val

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 22

<211> 107

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 22

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Lys Ser Trp

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Arg Ala Arg Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Val

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 23

<211> 107

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 23

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Lys Ser Trp

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Arg Ala Arg Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Val

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 24

<211> 107

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 24

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Lys Ser Trp

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Arg Ala His Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Val

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 25

<211> 107

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 25

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Arg Ala His Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Val

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 26

<211> 245

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Thr Gly Arg Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Pro Asn Thr Gly Gly Asp Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile Gln Leu Thr Gln

130 135 140

Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

145 150 155 160

Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Arg Gln

165 170 175

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu

180 185 190

Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

195 200 205

Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

210 215 220

Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile Thr Phe Gly Gln Gly Thr

225 230 235 240

Arg Leu Glu Ile Lys

245

<210> 27

<211> 447

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Thr Gly Arg Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Arg Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Pro Asn Thr Gly Gly Asp Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 28

<211> 214

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 28

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Arg Ala Arg Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Val

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 29

<211> 447

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 29

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Thr Gly Arg Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Pro Asn Thr Gly Gly Asp Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 30

<211> 214

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 30

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 31

<211> 122

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 31

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ala Leu Ile Asn Pro Tyr Lys Gly Val Thr Thr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 32

<211> 107

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys

100 105

<210> 33

<211> 244

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 33

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Cys Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr

165 170 175

Lys Gly Val Thr Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr

210 215 220

Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val

225 230 235 240

Thr Val Ser Ser

<210> 34

<211> 10

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 34

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 35

<211> 701

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 35

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Thr Gly Arg Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Arg Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Pro Asn Thr Gly Gly Asp Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

450 455 460

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

465 470 475 480

Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys

485 490 495

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu

500 505 510

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr

515 520 525

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

530 535 540

Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Cys Gly Thr Lys

545 550 555 560

Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

565 570 575

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe

595 600 605

Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu

610 615 620

Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Thr Thr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn

645 650 655

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

660 665 670

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe

675 680 685

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

690 695 700

<210> 36

<211> 701

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Thr Gly Arg Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Pro Asn Thr Gly Gly Asp Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

450 455 460

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

465 470 475 480

Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys

485 490 495

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu

500 505 510

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr

515 520 525

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

530 535 540

Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Cys Gly Thr Lys

545 550 555 560

Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

565 570 575

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe

595 600 605

Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu

610 615 620

Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Thr Thr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn

645 650 655

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

660 665 670

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe

675 680 685

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

690 695 700

Claims

1. A TROP2 antibody comprising a heavy chain variable region and a light chain variable region, wherein the CDRs of the heavy chain variable region and the CDRs of the light chain variable region are any one of:

(1) The amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO.1, the amino acid sequence of CDR2 is shown as SEQ ID NO.2, the amino acid sequence of CDR3 is shown as SEQ ID NO.3, the amino acid sequence of CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is shown as SEQ ID NO.5, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 6;

(2) The amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO.7, the amino acid sequence of CDR2 is shown as SEQ ID NO.8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3; the amino acid sequence of CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is shown as SEQ ID NO.13, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 15;

(3) The amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO.7, the amino acid sequence of CDR2 is shown as SEQ ID NO.8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3; the amino acid sequence of CDR1 of the light chain variable region is shown as SEQ ID NO.12, the amino acid sequence of CDR2 is shown as SEQ ID NO.13, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 15;

(4) The amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO.7, the amino acid sequence of CDR2 is shown as SEQ ID NO.8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3; the amino acid sequence of CDR1 of the light chain variable region is shown as SEQ ID NO.12, the amino acid sequence of CDR2 is shown as SEQ ID NO.14, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 15.

2. The TROP2 antibody of claim 1, wherein said heavy chain variable region has the amino acid sequence set forth in SEQ ID No.16 and said light chain variable region has the amino acid sequence set forth in SEQ ID No. 17;

3. The TROP2 antibody of claim 1 or 2, wherein the Fc fragment of the heavy chain is the Fc fragment of a human or humanized antibody that is IgG1, igG2, igA, igE, igM, igG, 4 or IgD.

4. The TROP2 antibody according to claim 1 or 2, wherein the heavy chain of said antibody has the amino acid sequence shown in SEQ ID No.27 and the light chain has the amino acid sequence shown in SEQ ID No. 28; or the heavy chain of the antibody has an amino acid sequence shown as SEQ ID NO.29, and the light chain has an amino acid sequence shown as SEQ ID NO. 30.

5. A single chain antibody, fab antibody, minibody, chimeric antibody, whole antibody immunoglobulin IgG1, igG2, igA, igE, igM, igG, igD, bispecific antibody or multispecific antibody comprising the TROP2 antibody of any one of claims 1-4.

6. Bispecific antibody that binds TROP2 and CD3, comprising:

a first domain that binds to trophoblast cell surface antigen 2,

And, a second domain that binds the T cell surface antigen CD3,

The first domain comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are the heavy chain variable region and the light chain variable region of the TROP2 antibody according to claim 1 or 2.

7. The bispecific antibody binding to TROP2 and CD3 according to claim 6, characterized in that said first domain is 2 complete light chain-heavy chain pairs linked by disulfide bonds, the amino acid sequences of the heavy and light chains of which are the amino acid sequences of the heavy and light chains of the TROP2 antibody according to claim 4.

8. The bispecific antibody binding to TROP2 and CD3 according to claim 6 or 7, characterized in that the heavy chain variable region of said second domain has the amino acid sequence shown in SEQ ID No.31 and the light chain variable region has the amino acid sequence shown in SEQ ID No. 32.

9. The bispecific antibody binding to TROP2 and CD3 according to claim 8, characterized in that the light chain variable region and the heavy chain variable region of said second domain are linked by a linker peptide into a single chain antibody having the amino acid sequence shown in SEQ ID No. 33.

10. A bispecific antibody binding to TROP2 and CD3 according to any one of claims 6, 7, 9, characterized in that said second domain comprises 2 single chain antibodies, said bispecific antibody being of symmetrical structure linked by any one of the following means:

(2) The N-terminal of the 2 single-chain antibodies of the second domain are respectively connected with the C-terminal of the 2 heavy chains of the first domain through connecting peptides.

11. The bispecific antibody binding to TROP2 and CD3 according to claim 10, characterized in that the amino acid sequence of said connecting peptide is (GGGGX) n, wherein X is Gly or Ser, n is a natural number from 1 to 4.

12. The bispecific antibody binding to TROP2 and CD3 according to claim 11, characterized in that the amino acid sequence of said connecting peptide is shown in SEQ ID No. 34.

13. The bispecific antibody binding to TROP2 and CD3 according to any one of claims 6, 7, 9, 11, 12, characterized in that the heavy chain of said first domain has the amino acid sequence shown in SEQ ID No.35 or 36 after being linked to said second domain via a linker peptide, and the light chain has the amino acid sequence shown in SEQ ID No.28 or 30.

14. The bispecific antibody binding to TROP2 and CD3 according to claim 13, characterized in that the heavy chain of said first domain has the amino acid sequence shown in SEQ ID No.35 after being linked to said second domain via a linker peptide, the light chain has the amino acid sequence shown in SEQ ID No.28, or the heavy chain of said first domain has the amino acid sequence shown in SEQ ID No.36 after being linked to said second domain via a linker peptide, the light chain has the amino acid sequence shown in SEQ ID No. 30.

15. A nucleic acid molecule encoding a TROP2 antibody according to any one of claims 1 to 4, or encoding a bispecific antibody binding TROP2 and CD3 according to any one of claims 6 to 14.

16. A biological material comprising the nucleic acid molecule of claim 15, said biological material comprising recombinant DNA, expression cassette, vector, host cell.

17. A method of producing a TROP2 antibody according to any one of claims 1 to 4 or a bispecific antibody binding TROP2 and CD3 according to any one of claims 6 to 14, comprising: introducing a nucleic acid encoding said antibody into a host cell to obtain a host cell stably expressing said bispecific antibody; culturing host cells, and separating and purifying to obtain the antibody.

18. Use of a TROP2 antibody according to any one of claims 1 to 4 or a bispecific antibody according to any one of claims 6 to 14 that binds TROP2 and CD3 or a nucleic acid molecule according to claim 15 or a biomaterial according to claim 16, as follows:

(1) Use in the manufacture of a medicament for the diagnosis, prevention or treatment of a tumor expressing TROP 2; wherein the tumor is gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, esophageal cancer, pancreatic cancer, head and neck cancer, ovarian cancer or intrauterine mucosa serous papilla cancer;

(2) Use in the manufacture of a medicament for killing a TROP2 expressing tumor cell; wherein the tumor cells are gastric cancer cells, cervical cancer cells, breast cancer cells, lung cancer cells, prostate cancer cells, colon cancer cells, esophagus cancer cells, pancreas cancer cells, head and neck cancer cells, ovarian cancer cells or intrauterine mucosa serous papillary carcinoma cells;

(3) The application in preparing TROP2 and/or CD3 detection reagent;

(4) Use in the manufacture of a medicament for CAR-T therapy;

(5) Use in the preparation of immunotoxins or labeled antibodies.

19. A multispecific antibody, fusion protein, immunotoxin, drug or detection reagent comprising a TROP2 antibody of any one of claims 1 to 4 or a bispecific antibody that binds TROP2 and CD3 of any one of claims 6 to 14.